KR0127776B1 - Novel protease and its cdna sequence - Google Patents

Abstract

The invention relates to a novel cDNA sequence encoding a thrombin-like serine protease obtainable from the venom gland of a Korean viper, Salmosa (Agkistrodon halys brevicaudus), and to an amino acid sequence translated therefrom.

Description

CDNA sequences of novel proteases and proteins derived therefrom

Figure 1 is a schematic diagram showing the manufacturing process of the venom gDNA library of the Korean salsa.

Figure 2 (A) shows the DNA sequence of a PCR product derived from the cDNA library and the amino acid sequence translated therefrom.

Figure 2 (B) shows the comparison of the amino acid sequence of the PCR product and other snake venom serine protease.

3 is a photograph showing the results of the screening of the cDNA library.

FIG. 4 (A) schematically shows a DNA sequence of Halib.

4 (B) shows the cDNA sequence of halibin.

FIG. 4 (C) shows the amino acid sequence translated from the cDNA sequence of halibin disclosed in FIG. 4 (B).

5 shows a comparison of the amino acid sequence of halibin with the amino acid sequence translated from the DNA sequence of the PCR product.

FIG. 6 shows a comparison of the amino acid sequences of halibins and known snake venom serine proteolytic enzymes.

The present invention relates to novel protease cDNA sequences and amino acid sequences derived therefrom. More specifically, the present invention relates to a cDNA sequence of a novel thrombin-like serine protease isolated from a venom gland of Korean salmosa snake (Agkistrodon halys brevicaudus) and an amino acid sequence translated therefrom. .

Thrombosis and hemostasis have long been of major medical interest. For the treatment of thrombosis, urokinase, streptokinase, and tissue-type plasminogen activators have been studied and put to practical use (HR Lijnen). et al., Thromb. haemost., 66: 88 (1991)). In recent years, however, efforts have been made to extract and extract active ingredients from natural products, which have separated substances that can be used as therapeutic agents for blood clots from leeches, vampire bats, and snake venom (Roy T. Sawyer, Bio / Technology, 9). : 513 (1991); USP 4,568,545; Stephen J. Gardell et al., J. Biol. Chem., 264: 17947 (1989); J. Meier and K. Stocker, Toxicology, 21: 171 (1991).

At this level, snake toxins, which are known to affect the human hemostatic system, have been studied in more detail, and a number of toxins that act on hemostatic action have been isolated and tested. Many of these materials have been applied to basic research, diagnosis, and treatment. In particular, thrombin that cuts fibrinopeptide into fibrinogen to convert fibrinogen into fibrin Studies on various types of snake venom related to similar proteases have been published (J. Meier and K. Stocker, Toxicology, 21: 171 (1991)). At present, 23 kinds of enzymes are known, and in 4 cases, the amino acid sequence of the whole enzyme is also known.

Known in detail for their enzymatic properties, the thrombin-like proteolytic enzyme batroxobin isolated from the venom of Bothrops atrox moojeni, known as the Lance head snake, is called fibrino of fibrigen. It has the property of cleaving only peptide A. Upon treatment with a small amount of bartroxobin, fibrinogen is converted to fibrin I (Des-A-fibrin) and rapidly degraded by plasmin, resulting in lower fibrinogen concentration in the blood. In addition, it induces the production of plasminogen activator and promotes the decomposition of blood clots, but induces coagulation in large amounts of treatment. In the case of using the former property, a defibrinogenating drug called Defibrase is used as a hemostatic agent under the name Reptilase (Refer to J. Meier). and K. Stocker, Medical Use of Snake Venom Proteins, CRC Press, 136 (1990)).

On the other hand, there are 14 kinds of snakes in Korea, and among them, only three species of Agkistrodon are known to have toxins (KY Nah, Korean J. Surgery, 17: 13 (1975); HK) Gloyd Proc, Biol. Soc., Washington, 85: 577 (1971). Recently, 39,200 daltons of thrombin-like proteolytic enzymes have been isolated from venom from Agristrodon halys brevicaudus, commonly referred to as salsa. The enzyme is a glycoprotein and consists of 323 amino acids and contains no significant amounts of glutamic acid and aspartic acid, but other biochemical activities have not been elucidated (see J. Sun, Y. Ren, Z. WU, Zhongguo Yike Daxue Xuebao, 16: 276 (1987); Chem. Abstr., 108: 90629n (1988)). Among these, 33,000 daltons and 51,000 daltons of fibinolytic serine protease have been reported from the venom venom (KH Chung et al., Thromb. Haemost. THHADQ, 65: 953 (1991). )).

Accordingly, the inventors of the present invention have found that the cDNA of proteolytic enzymes similar to thrombin cloned from the snake venom cDNA library of Korean Salmonosa and the amino acid sequence translated therefrom are novel and completed the present invention.

After all, the main object of the present invention is to provide a cDNA sequence of a novel thrombin-like protease isolated from the venom of the Korean Salmosaci agstrostrodon Halis Brevicaudus species and the amino acid sequence translated therefrom.

Hereinafter, the present invention will be described in more detail.

The inventors used a PCR product as a probe from oligonucleotide primers and cDNA libraries of venom glands for amino acid sites well conserved in serine-based proteolytic enzymes isolated from snake venom. The cDNA coating was cloned. The cloned cDNA has an open reading frame of 837 nucleotides that coats 279 amino acids, and the presumed amino acid sequence is a signal peptide consisting of 45 amino acids and a serine proteolysis of a mature enzyme of 234 amino acids. It was confirmed to be separated by an enzyme (hereinafter referred to as halibin for convenience). Since halibin has an estimated molecular weight of 25.7 kD from the amino acid sequence and one N-glycosylation site, it is expected that glycoprotein having a higher molecular weight in the venom solution of salsa is expected to be a coin point from the amino acid component. (pI) was estimated to be 5.68.

On the other hand, from the information search results of the protein, it was confirmed that this serine proteolytic enzyme has a similarity on the amino acid sequence of 64 to 71% with known thrombin-like proteases of snake venom. In addition, the novel protein of the present invention has the amino acid sequence of His, Asp, Ser, and its surroundings, and the amino acid sequence of the N-terminal region as well as the enzymes known in the art. It was confirmed that it was preserved. In addition, since all 12 cysteines conserved in the entire amino acid sequence of enzymes belonging to this class are well conserved, the novel protein of the present invention may have similar proteolytic activity to thrombin. It was expected.

The cDNA sequence of a novel thrombin-like proteolytic enzyme isolated from the venom of the Korean Salmosa virus aquistrodone Halis Brevikadus species of the present invention is established in recombinant E. coli, yeast, baculovirus / insect cells and animal cells. Can be expressed by the expression system, the proteolytic enzymes produced in large quantities in this way will be useful in the preparation of thrombosis therapeutics and hemostatic agents.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1

Preparation of venom gDNA library of Korean salsa

The venom gland tissue of Korean salsa agstrostrodon Halis Brevicaudus was isolated. The venom gland was a small bean-sized tissue, and at least five were used to isolate a sufficient amount of RNA. Isolation of total RNA was carried out using a method of high speed centrifugation under a CsCl cushion using a known guanidine isothiocyanate. Poly (A) + RNA was isolated from the total RNA using twice the oligo (dT) -cellulose column and then the first and second strand cDNAs were prepared using reverse transcriptase, RNase H and E. coli DNA polymerase I. Was prepared (Sambrook et al., Molecular Cloning, Znd Ed., Cold Spring harbor laboratory (1989)). An appropriate adapter was attached to the prepared cDNA, then inserted into lambda ZAPII (Stratagene, USA), and the library was completed through in vitro packaging. The cDNA library was assayed to contain 10 ⁶ independent plaques. The process of producing the venom glands cDNA library of Korean salsa is shown in FIG.

Example 2

Preparation of the Probe

To select probes for screening thrombin-like proteases using the cDNA library prepared in Example 1, oligo nucleotides for the well-conserved amino acid sites in serine-based proteases of snake venom were synthesized: That is, as the 5'-primer, an oligonucleotide having a 5'-GTIATIGGIGGNGA (T / C) GA (A / G) TG-3 'sequence based on VIGGDEC of the common amino acid sequence of the N-terminal region of the snake venom is selected. Used; The 3'-primer used a degenerate oligonucleotide primer having a 5'-TGIGCIGCIGTNA (A / G) NACCCA-3 'sequence based on the amino acid sequence WVLTAAH around histidine, one of the active sites of the serine protease. (Wherein I is the inosine rate; N represents each nucleotide of A, G, C, T).

Using the cDNA library prepared in Example 1 as a template, using the primers and Taq DNA polymerase, 25 polymerase chain reactions were performed at 95 ° C. for 1 minute, 48 ° C. for 2 minutes, and 72 ° C. for 1 minute. Polymerase chain reaction (PCR) was performed to obtain 120 bp PCR product. The obtained PCR product was subcloned into plasmid pCRII (Invitrogen, USA) to determine DNA sequence (Fig. 2 (A)).

The amino acid sequence derived from the DNA sequence (PCR 120) was found to have 65-72% homology with the known serine protease, ie FLAVOXOBIN isolated from BATROXOBIN and Trimeresurus flavaviridis isolated from Bothropsatrox moojeni. (Fig. 2 (B)). In Figures 2 (A) and 2 (B), the underlined portion is the sequence of DNA derived from the PCR primer, and the box portion is the sequence of the DNA used for cDNA library screening.

Therefore, for screening the actual cDNA library, the base sequence 5'-CGTTCCCTTGTTGTCTTGTTTAACTCCAGCG-3 'corresponding to the middle portion of the 120bp PCR product was used.

Example 3

Screening of cDNA Libraries

The cDNA library prepared in Example 1 was plated on E. coli XL-1 Blue to obtain a total of 1.2 × 10 ⁵ plaques. The plaques were transferred to the nitrocellulose membrane twice, and then the membrane was treated with denatured solution (0.5N NaOH, 1.5M NaCl) for 2 minutes, neutralized solution (0.5M Tris HCl (pH 7.4), 1.5M NaCl) for 5 minutes. , Washed for 30 seconds in 2 x SSC. The membranes were treated under vacuum at 80 ° C. for 2 hours and then prehybridized at 68 ° C. for 3 hours in a solution containing 5 × SSC, 20 mM sodium phosphate (pH 6.5), 3% SDS, 100 μg / ml salmon sperm DNA. (prehybridization). The probe selected in Example 2 was end-labeled with [γ- ³² P] ATP and added to the prehybridization solution at a concentration of 1 × 10 ⁶ cpm / ml, followed by hybridization at 53 ° C. for 6 hours. It was. The membrane was then washed at 53 ° C. for 1 hour in a solution containing 3 × SSC, 3% SDS, 10 mM sodium phosphate (pH 6.5), then at 53 ° C., 1 hour in a solution containing 1 × SSC, 1% SDS. Washed during. The membranes were exposed to X-ray film (Agfacurix) at −70 ° C. for 24 hours under an intensifying screen. As a result, in the duplicate of the X-ray film, 11 positive clones with spots formed at the same position were found (FIG. 3). The photograph of FIG. 3 shows the result of hybridizing phage plaques transferred from the same plate to two nitro cellulose membranes and then exposing them to an X-ray film. Next, the result of exposure to the X-ray film is shown, showing a positive result in which a spot appears at the same position of each film. Low dilution multiples were also plated and the secondary and tertiary screenings were repeated in the same manner as above, resulting in independent plaques.

Each clone was subcloned into the pBluescript vector (Stratagene, USA), and the restriction enzyme treatment resulted in an insert size of 0.5 kb to 2.1 kb. Each clone determined a partial sequence, and it was confirmed that 11 clones contained a part of the same gene, which determined the sequence of the entire longest 2.1kb clone.

Example 4

Determination of DNA Sequence

The 2.1 kb clone was subjected to a dideoxy termination method using Sequinase (USB, USA) (Sambrook et al. Molecular Cloning, 2nd Ed., (1989)) for the primer and cDNA sequences on the vector. The sequences were determined on both sides using the primers derived. Determination of the sequence was performed centrally on the site including the entire open reading frame in 2.1 kb of the total insert, resulting in a sequence of 1003 bp. The determined sequence includes 49 bp 5'-untranslation region, 837 bp open reading frame (ORF), and 114 bp 3'- untranslated region (Fig. 4 (A)).

Example 5

DNA sequence analysis

The DNA sequence determined that the cDNA had an open reading frame of 837 nucleotides, which coated 279 amino acids. The protein, consisting of 279 amino acids, consisted of a 45 amino acid signal peptide and 234 amino acids as a mature enzyme. It was expected to exist (figure 4 (B)). Since the enzyme described has a molecular weight of 25.7 kD from the amino acid sequence and has one possible N-glycosylation region, it is expected to be a glycoprotein with a higher molecular weight in the venom solution of Salsa, and isoelectric point (pI) from the amino acid component. This was estimated at 5.68.

When comparing this clone (HALYBIN) with the DNA sequence (PCR 120) and amino acid sequence of the 120 bp PCR product obtained in Example 2, it was found that the three nucleotides and the corresponding three amino acids were different (FIG. 5). . From these results, it could be expected that there are very similar serine proteolytic enzymes with different amino acid sequences in the salsa venom solution. In Fig. 5, the asterisk denotes a conserved amino acid; ($) Indicates an active part of them; (#) Denotes conserved cysteine residues, respectively.

On the other hand, from the information search results of the protein using Swiss-Port, it was confirmed that the serine proteolytic enzyme has a similarity in the amino acid sequence of 64 to 71% with known thrombin-like proteases of snake venom (Sixth Degree). In addition, the novel protein of the present invention is composed of His, Asp, Ser (denoted by $ in FIG. 6) and its surrounding amino acid sequence, and the amino acid sequence of the N-terminal region, which constitute the active site of the serine protease. It is confirmed that it is well conserved, as is the known enzymes. In particular, it was found that all 12 cysteines (denoted by # in FIG. 6) conserved throughout the amino acid sequence of enzymes belonging to this class are well conserved. Therefore, the novel protein of the present invention is also expected to have proteolytic activity similar to thrombin, so it was decided to call it Halin.

As described in detail above, the present invention provides a cDNA sequence of a novel thrombin-like protease isolated from the venom gland of Agki strodon-Hallys Brevikadus species, a Korean salsa, and an amino acid sequence derived therefrom. The cDNA sequence of a novel thrombin-like protease isolated from the venom gland of Agkistrodon Halis Brevikadus species, a Korean salmosa of the present invention, is established in recombinant E. coli, yeast, baculovirus / insect cells and animal cells. The protein hydrolase produced in large quantities in this manner may be usefully used for the preparation of a thrombosis therapeutic agent and a hemostatic agent.

Claims (4)

CDNA of a thrombin-like proteolytic enzyme having all or part of the following sequence isolated from the venom gland of the Korean salmolog Agkistrodon halys brevicaudus: A probe having the following sequence for use in screening a library of cDNAs according to claim 1: 5'-CGTTCCCTTGTTGTCTTGTTTAACTCCAGCG-3 ' An amino acid sequence having all or part of the following sequence derived from the cDNA disclosed in claim 1: 5 10 15 20 25 30 1 M A L I R V L A N L L I L Q L S Y D L L H Y T V C N I W V I 31 A S E T N L K T A Q K S S E L V V G G D E C N I N E H R S L 61 V V L F N S S G L I C S G T L I N Q E W V L T A A H C D S K 91 N F Q M L F G V H S K K I L N E D E Q T R D P K E L F I C P 121 N K K K D D E L D K D I M L I R L D S P V S N S E H I A P L 151 S L P S S S P T V D S V C R I M G W G T I K P A D E T Y P D 181VPHCANINILDHTVCRAAYPVLLAGSSTLC 211AGTQQGGKDTCVGDSGGPLICNGQIQGIVS 241WGAHPCGQGSKPGVYTKVFDHLDWIKSIIA 271GNTAVTCPP * A thrombin-like proteolytic enzyme isolated from the venom gland of Agkistrodon halys brevicaudus, a Korean salmosa having the amino acid sequence of claim 3.